4024
Short Report
Ancestral centriole and flagella proteins identified by
analysis of Naegleria differentiation
Lillian K. Fritz-Laylin and W. Zacheus Cande*
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
*Author for correspondence ([email protected])
Accepted 25 August 2010
Journal of Cell Science 123, 4024-4031
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.077453
Journal of Cell Science
Summary
Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton
de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a
cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout
Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism,
signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55
genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late). The early
set includes genes encoding nearly every known conserved centriole component, as well as eight previously uncharacterized, highly
conserved genes. The human orthologs of at least five genes localize to the centrosomes of human cells, one of which (here named
Friggin) localizes specifically to mother centrioles.
Key words: Naegleria, Assembly, Centriole, Evolution, Flagella
Introduction
Naegleria gruberi grows as an amoeba without flagella, centrioles
or even cytoplasmic microtubules; it relies on an actin-based
cytoplasmic cytoskeleton for chemotaxis and motility, and its
mitotic spindle is contained within an intact nuclear envelope
(Fulton, 1970; Fulton, 1977; Fulton and Dingle, 1971). However,
when exposed to stressors such as changes in temperature or
nutrient availability, Naegleria rapidly differentiates into a
flagellate, forming a complete cytoplasmic microtubule
cytoskeleton from scratch (Fig. 1) (Fulton and Dingle, 1967). This
differentiation occurs synchronously – approximately 90% of cells
assemble basal bodies (structures equivalent to centrioles) within
a 15-minute window, followed by flagella approximately 10
minutes later (Fig. 1) (Fulton and Dingle, 1971). Although
Naegleria assembles basal bodies de novo, protein incorporation
occurs in the same order as that occurring during assembly of
human centrioles (Fritz-Laylin et al., 2010a). The evolutionary
distance of Naegleria from animals means that genes shared
between Naegleria and humans were probably present in the
ancestor of all eukaryotes (Cavalier-Smith, 2002; Ciccarelli et al.,
2006) (for a review, see Fritz-Laylin et al., 2010b). Thus, Naegleria
differentiation affords a unique opportunity to study ancestral
features of centriole and flagellum assembly.
Interphase animal cells contain numerous microtubules
emanating from microtubule organizing centers (MTOCs) called
centrosomes. Centrosomes contain centrioles that are primarily
composed of nine microtubule triplets, and the surrounding
amorphous pericentriolar material (PCM) that anchors cytoplasmic
microtubules. Centrioles are called basal bodies when they are
used to organize axonemes, the microtubule core of eukaryotic
cilia and flagella. These whip-like structures propel single-celled
organisms and move fluids within multicellular organisms.
Metazoan cells also have nonmotile cilia that function as ‘cellular
antennae’ by gathering information about the surrounding
environment using their varied signaling receptors (Marshall and
Nonaka, 2006).
Proteomic analyses indicate that centrosomes and basal bodies
contain many of the same proteins, a large number of which are
thought to be functional components of centrioles (Andersen et al.,
2003; Keller et al., 2005; Kilburn et al., 2007). However, only a
handful of these proteins have been characterized functionally
(Strnad and Gönczy, 2008). This is in part due to the technical
difficulties associated with studying centriole assembly in most
organisms. First, new centrioles usually assemble in association
with a mature centriole, hindering proteomic characterization of
assembly intermediates. Second, centriole assembly is usually tied
to the cell cycle, rendering it difficult to distinguish centriolespecific genes from other induced cell cycle genes. And finally, de
novo assembly (where centrioles are built in the absence of
preexisting ones) usually occurs in a single cell or embryo, making
proteomic or microarray-based approaches unfeasible. Here, we
used the synchronous de novo basal body assembly pathway of
Naegleria to overcome these technical roadblocks and identify
genes used specifically for building basal bodies and flagella.
Results and Discussion
Flagella and basal body gene transcripts are induced with
different kinetics
We isolated total RNA at 20-minute intervals during Naegleria
differentiation (at 0, 20, 40, 60 and 80 minutes; Fig. 1) from three
biological replicates (supplementary material Fig. S1A). The
relative abundance of transcripts from each time-point was
quantified using custom full-genome Naegleria DNA microarrays.
Approximately 24% of Naegleria genes are induced at least
twofold, and an additional 39% are reduced by at least 50% during
the amoeba-to-flagellate transition (4065 and 6484 genes,
respectively; P<0.01, after correction for multiple testing).
Differentially regulated genes include those involved in stress
Finding centriole genes with Naegleria
4025
Centriole-enriched gene cluster
Journal of Cell Science
Fig. 1. Selected events during differentiation of Naegleria. Each of the three
curves represents the percentage of cells with visible flagella during one
differentiation replicate used for microarray analysis; the time-points collected
are indicated with an asterisk (*). Important events during assembly of the
Naegleria basal body are indicated.
responses (including Hsp20 and Hsp 90; data not shown) and core
metabolism (including glycolysis, Krebs cycle and pyruvate–acetate
metabolism; data not shown), as well as cytoskeletal components.
Only a fraction of the thousands of induced genes are likely to
be microtubule related. To aid in our search for uncharacterized
and evolutionarily conserved centriole proteins, we focused on
genes found in Naegleria and other flagellates but missing in nonflagellated organisms [the flagellar motility (FM) gene set (FritzLaylin et al., 2010b)]. FM members include genes specific to
basal bodies and flagella but exclude genes such as that encoding
a-tubulin that are also used by organisms without flagella. To
permit analysis of general microtubule proteins involved in basal
body and flagella formation, we added Naegleria homologs of
known microtubule cytoskeleton proteins (Fritz-Laylin et al.,
2010b). Finally, we also added 63 genes conserved in organisms
that undergo amoeboid movement and missing in organisms that
do not undergo amoeboid locomotion [the amoeboid motility
(AM) gene set] (Fritz-Laylin et al., 2010b), to serve as a specificity
control.
Overall, 78% of the FMs and 60% of the AMs have at least
twofold induction or repression, respectively (P<0.01, after
correction for multiple testing), providing large-scale confirmation
of previous evidence that Naegleria differentiation is controlled at
the transcriptional level (Lai et al., 1988; Levy et al., 1998). We
next investigated whether the timing of gene expression was linked
to function.
Cluster analysis of the expression data for these 310 genes (the
AM and FM gene sets, and Naegleria homologs of known
microtubule genes) resulted in five major gene clusters (A–E; Fig.
2). Clusters A and C consist primarily of genes found in the FM
gene set and have increased expression during differentiation.
However, the genes in cluster A reach peak expression levels by
20 minutes and begin decreasing in expression by 40 minutes,
whereas the expression of genes in cluster C peaks by 40 minutes
and remains high through to 80 minutes. Manual inspection
revealed that the cluster with earlier expression contains many
known centriole genes (Table 1), whereas the later expression
cluster contains flagella genes (supplementary material Table S1).
The general induction of basal body genes before flagella genes
agrees with the fact that Naegleria assembles its basal bodies
before it assembles its flagella (t55 and t65 minutes, respectively)
(Fig. 1) (Fritz-Laylin et al., 2010a; Fulton and Dingle, 1971).
The 55 genes found in the centriole gene cluster include Naegleria
homologs of seven genes whose products are thought to be required
for assembly of the centriole or basal body: e-, d- and h-tubulin,
SAS-4 (CPAP), SAS-6, centrin (Cen2) and POC1 (for references,
see Table 1). This set represents the majority of components shown
to be required specifically for centriole assembly that are conserved
outside animals (Carvalho-Santos et al., 2010; Hodges et al., 2010;
Strnad and Gönczy, 2008). Other core centriole genes not found in
the cluster either have not been identified in the Naegleria genome
(PLK4) or were not included in the microarray (BLD10).
The centriole-enriched gene cluster also encodes homologs of
microtubule nucleation factors [g-tubulin, GCP3 and GCP6
(Raynaud-Messina and Merdes, 2007)], as well as proteins required
for general microtubule functions, such as the microtubule-severing
protein katanin p60, which is known to localize to centrosomes
(Hartman et al., 1998). This gene set also includes several genes
encoding centrosome-localized proteins of unknown function, and
eight completely uncharacterized genes (Table 1).
Axonemal dyneins are large protein complexes containing light,
intermediate and heavy chains that slide microtubules past each
other to produce flagellar movement. Surprisingly, the centrioleenriched cluster includes nine axonemal dynein light and
intermediate chain homologs, as well as a homolog of kintoun
(PF13) that is required for assembly of dynein arm complexes
(Omran et al., 2008). By contrast, Naegleria dynein heavy chain
genes are expressed later along with other flagella-specific genes.
Assembly of dynein light and intermediate chain complexes can be
genetically uncoupled from assembly of the dynein heavy chain in
Chlamydomonas (Omran et al., 2008). Although there are many
possible reasons for the early expression of dynein light and
intermediate chains, but not heavy chains, it is possible that
Naegleria pre-assembles flagellar intermediate and light chain
dyneins before incorporating heavy chains, before flagellar
outgrowth.
Flagella-enriched gene cluster
The flagella-enriched gene cluster contains 82 genes
(supplementary material Table S1), including genes encoding
proteins used for transporting proteins to the base of the growing
flagellum (BBS components BBS1–BBS5 and BBS7–BBS9) and
within the flagellum to its growing tip (FLA3, kinesin 2, IFT20,
IFT52, IFT57, IFT80, IFT88, IFT122 and IFT140), as well as
structural components of the flagellum itself [including PF20 and
PF16, RSP4 and Rib72 (Pazour et al., 2005)]. This gene set also
includes 23 FM genes with homologs found in the Chlamydomonas
flagella proteome (Pazour et al., 2005) but which are otherwise
uncharacterized. Together, these data suggest that these proteins
are probably core components of eukaryotic flagella and therefore
prime candidates for future functional analyses.
To validate the putative flagellar components, we conducted a
proteomic analysis of Naegleria flagella. We purified the flagella of
~4⫻108 flagellate cells using low-speed centrifugation followed by
a sucrose step-gradient. The resulting sample contained flagella and
no visible cell bodies and comprised largely two proteins of a size
similar to that of a- and b-tubulin (supplementary material Fig. S1,
panel B), as is typical for clean flagellar preparations (e.g. Kowit and
Fulton, 1974). MUDPIT mass spectrometry analysis of the sample
identified 415 proteins (supplementary material Table S2).
Of the 82 genes in the flagellar-enriched gene cluster, 23 were
also identified in our proteomics analysis (supplementary material
Journal of Cell Science 123 (23)
Journal of Cell Science
4026
Fig. 2. Centriole- and flagella-enriched gene clusters. Each
row represents one gene, with columns representing expression
at the indicated time-point. Blue represents low expression,
and yellow represents high expression. The cladogram groups
genes based on similarity in expression across the five timepoints; five major clusters are indicated (A–E). Red and green
boxes on the right-hand side indicate membership of the AM
(amoeboid motility) and FM (flagellar motility) gene sets,
respectively. The graphs indicate relative gene expression –
using as examples centriole-enriched cluster gene (SAS-6) and
flagella-enriched cluster gene (IFT88). The data are plotted as
the means ± s.d. (n3).
Table S1), indicating that they are likely to be structural components
of the flagellum itself (in contrast to proteins that might be required
for flagellar function but are located within the cell body). Included
in this overlap are seven flagellar-associated proteins (FAPs), which
were identified in the proteomic analysis of Chlamydomonas
flagella (Pazour et al., 2005) but remain otherwise uncharacterized.
These proteins are therefore likely to be ancestral structural flagella
components. The Naegleria flagellar proteome also includes a
number of previously undescribed proteins (supplementary material
Table S2), some of which might represent uncharacterized flagellar
proteins.
Verification of putative centriole genes
The centriole-enriched gene cluster includes eight genes that have
not previously been localized or otherwise characterized, which
we refer to as ‘putative conserved centriole components’ (pCCCs;
Table 1). Because orthologs of all centrosome-localized pCCCs
can be found in a wide diversity of eukaryotes (supplementary
material Table S3), they were probably present in the eukaryotic
ancestor. To determine whether the pCCCs are likely to be centriole
components, we transiently expressed N- and C-terminally GFPtagged human orthologs of each pCCC in U2OS and HeLa human
cell lines and used antibodies recognizing g-tubulin to highlight
centrosomes. To the eight unknown gene products, we added one
whose homolog localizes to the base of the cilia of Caenorhabditis
elegans (B9D2) and one that has only very recently been
characterized in human cells (MOT52, also known as FOR20 or
BBC20) (Sedjai et al., 2010). Five of the ten tagged proteins
showed either diffuse cytoplasmic GFP or bright foci likely to be
inclusion bodies (data not shown). This nonspecific localization
neither confirms nor denies a possible centriole function. However,
the remaining five localized within or near centrosomes using both
N- and C-terminal GFP tags (Fig. 3A) and are described below.
First, MOT52 is found only in organisms with motile flagella
(Merchant et al., 2007), and its homolog (BBC20) was found in
the Tetrahymena basal body proteome (Kilburn et al., 2007).
Recently, the human ortholog (FOR20) was reported to localize to
pericentriolar satellites and to be involved in ciliary assembly
(Sedjai et al., 2010). FOR20 is predicted to have a FOP dimerization
domain (Pfam domain PF09398), required for the centrosomal
localization of the FOP protein (Mikolajka et al., 2006). In transient
transfections of both U2OS (Fig. 3A) and HeLa cells (data not
shown), GFP-tagged FOR20 localized to multiple foci near
centrosomes. A similar localization was reported using an antibody
Finding centriole genes with Naegleria
4027
Table 1. Centriole-enriched gene cluster
Class
BB, centriole assembly
Ortholog
-Tubulin
-Tubulin
Protein ID
44774
69007
Function
BB, centriole assembly
BB, centriole assembly
65724
BB, centriole assembly
SAS-6
68996
BB, centriole assembly
SAS-4 (CPAP)
61107
BB, centriole assembly
Centrin
44488
56351
BB, centriole assembly
POC1
-Tubulin
GCP6
GCP3
SSA11 (TTL13)
Katanin P60
-Tubulin
TBCE
FOR20 (MOT52)
POC12
PIBF
TTL1
MKS6 (CC2D2A)
MKS3 (meckelin)
B9D1
B9D2
MBO2
ODA6
33676
56069
61337
434
80835
63871
71268
51830
81169
52938
29577
73664
33283
77673
62841
52666
30379
62959
60431
IDA7
ODA6
DLC1
IDA4 (P28)
57343
79232
74922
82719
BOP5
63304
TCTEXT1
DLC1
MOT24
MOT45 (kintoun,
PF13)
FAP184
FAP215
FAP127
FAP57
Friggin (MOT37)
POC11
SSA3
Q6PH85
POC16
LRR6
TECT3
MOT39
?
PSK1
?
?
?
?
MOT50
29177
54720
32555
80717
BB, centriole assembly
MT nucleation
MT nucleation
MT nucleation
Tubulin tyrosine ligase
MT severing
MTs
MTs
Putative tubulin chaperone
Pericentriolar satellites, centrosome
Unknown
Unknown
Tubulin tyrosine ligase
Cilium, BB function
BB migration, membrane docking
Transition zone
Transition zone
Maintain direction of motility
Dynein intermediate chain 2,
axonemal
Dynein intermediate chain, axonemal
Dynein intermediate chain, axonemal
Dynein light chain, axonemal
Dynein light intermediate chain,
axonemal
Dynein light intermediate chain,
axonemal
Dynein light chain, axonemal
Dynein light chain, axonemal
Dynein light chain, axonemal
Axonemal dynein complex assembly
2066
66643
44967
61313
61232
70454
68814
49668
62107
31069
65759
72718
73058
78247
82958
48624
29264
81040
54684
71996
Unknown
Nucleotidase
Unknown
Unknown
Mother centriole localization
Centrosome localization
Centrosome localization
Unknown
Unknown
Unknown
Unknown
Unknown
Dual-specificity phosphatase
Nucleotide kinase
High-mobility-group protein
Lipid synthesis
HUS-1-like protein
BTG-domain protein
Chymotrypsin-like protein
Chymotrypsin-like protein
-Tubulin
Journal of Cell Science
MT-specific centrosome
function
BB, centriole, centrosome
localization
BB-specific function
Flagellar dynein complex
Putative flagellar function
Previously uncharacterized
(pCCCs)
Other
References
(Dutcher et al., 2002)
(Dutcher and Trabuco, 1998; Garreau de Loubresse et al.,
2001; O’Toole et al., 2003)
(Basto et al., 2006; Kirkham et al., 2003; Kleylein-Sohn et
al., 2007; Leidel and Gonczy, 2003; Pelletier et al.,
2006)
(Culver et al., 2009; Dammermann et al., 2004; Leidel et
al., 2005; Nakazawa et al., 2007; Peel et al., 2007;
Pelletier et al., 2006; Rodrigues-Martins et al., 2007;
Strnad et al., 2007)
(Basto et al., 2006; Kirkham et al., 2003; Kleylein-Sohn et
al., 2007; Leidel and Gonczy, 2003; Pelletier et al.,
2006)
(Baum et al., 1986; Koblenz et al., 2003; Kuchka and
Jarvik, 1982; Salisbury et al., 2002; Taillon et al., 1992;
Winey et al., 1991)
(Keller et al., 2009; Pearson et al., 2009)
(Raynaud-Messina and Merdes, 2007)
(Raynaud-Messina and Merdes, 2007)
(Raynaud-Messina and Merdes, 2007)
(Hammond et al., 2008)
(Hartman et al., 1998)
(Dutcher, 2001)
(Dutcher, 2001)
(Sedjai et al., 2010)
(Keller et al., 2005)
(Lachmann et al., 2004)
(Wloga et al., 2008)
(Gorden et al., 2008)
(Dawe et al., 2007)
(Williams et al., 2008)
(Williams et al., 2008)
(Tam and Lefebvre, 2002)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Harrison et al., 1998; Kagami et al., 1998)
(Pazour et al., 2005)
(Merchant et al., 2007)
(Omran et al., 2008)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
(Pazour et al., 2005)
This study
This study
This study
The 55 genes in the centrosome-enriched cluster are organized by function (or predicted function), followed by ortholog gene and/or protein names, with appropriate
references in the last column. ?, no identifiable ortholog; BB, basal body; MT, microtubule. Sequence information for Naegleria protein IDs is available at
www.jgi.doe.gov/naegleria.
Journal of Cell Science
4028
Journal of Cell Science 123 (23)
Fig. 3. Human orthologs of pCCCs localize to centrosomes,
including the mother centriole protein Friggin. GFP-tagged
human orthologs of the indicated pCCC are shown in green
after transient transfection of U2OS cells. (A)Centrosomes
(arrows) are stained using an antibody against g-tubulin (red),
and DNA is shown in blue. (B)Centrosomes (arrows) are
stained with an antibody against g-tubulin (blue), mother
centrioles with an antibody against cenexin (red),and DNA is
shown in white. Scale bars:5m.
against FOR20 (Sedjai et al., 2010), thus validating our GFP
tagging approach.
Second, B9D2 contains a B9 domain. The C. elegans homolog,
TZA-1, localizes to the transition zone at the base of the cilium
(Williams et al., 2008). Although the B9 domain has no known
function, it is found in several proteins known to be localized to
the centriole and/or basal body, including MKS1 (Dawe et al.,
2007). Human B9D2 localized to centrosomes of U2OS cells,
along with scattered foci throughout the cytoplasm (Fig. 3A).
Third, POC11 shows good conservation in many eukaryotes but
has no identifiable domains other than a coiled-coil region.
Localization of the human homolog (CCDC77) resulted in bright
punctate spots within centrosomes of both U2OS and HeLa cells
(Fig. 3A and data not shown, respectively), suggesting that POC11
represents a new family of centrosome proteins.
Fourth, SSA3 was so named for its predicted function in both
motile and nonmotile flagella [SSA stands for ‘sensory, structural
and assembly’ (Merchant et al., 2007)]. SSA3 has a conserved
central region containing an ‘ELMO/CED12’ domain (Pfam domain
PF04727), found in proteins that facilitate cytoskeletal
rearrangements (Gumienny et al., 2001). SSA3–GFP-expressing
cells contained diffuse cytoplasmic GFP, as well as centrosomal
GFP in a small percentage (4%) of transfected cells that also
displayed relatively small g-tubulin foci (Fig. 3A). As g-tubulin
foci vary in size during the mammalian cell cycle (with small foci
at G1- and early S-phases), SSA3 might localize to centrosomes in
a cell-cycle-dependent manner.
Fifth, Friggin was originally named MOT37 for its predicted
function in motile flagella (Merchant et al., 2007) and contains
leucine-rich repeats (LRRs), which typically mediate protein–
protein interactions [Pfam clan CL0022 (Kobe and Deisenhofer,
1994)]. Unexpectedly, the 542-residue human ortholog of MOT37
localized to only one of two g-tubulin foci in both U2OS (Fig. 3A)
and HeLa cells (data not shown), suggesting that it might be a
component specific to either mature or immature centrioles.
Centrioles develop over two cell cycles, acquiring the basic
nine-triplet pinwheel structure during the first cell cycle and various
appendages that allow it to function as a basal body for axonemal
assembly during the second cycle. Several gene products have
been shown to be involved in the assembly of appendages
(Chang et al., 2003; Gromley et al., 2003; Lange and Gull, 1995;
Mogensen et al., 2000; Ou et al., 2002), only one of which,
e-tubulin (Chang et al., 2003), is conserved outside animals and
likely to be ancestral to all extant eukaryotes.
To investigate whether MOT37 is a component of either mother
or daughter centrioles, we expressed GFP–MOT37 and stained
cells with an antibody recognizing the mother centriole component
cenexin (Lange and Gull, 1995). GFP–MOT37 consistently
colocalized with cenexin (Fig. 3B), indicating that MOT37 is a
mother-centriole-specific protein that we predict is involved in the
developmental transition from immature to mature centrioles.
Because MOT37 probably represents a second ancestral mother
centriole protein, we have named this eukaryotic protein family
‘Friggin’ after Frigg, the Norse goddess of motherhood.
Concluding remarks
Understanding how centrioles and flagella assemble and function
requires a full inventory of components. Previous studies have
Journal of Cell Science
Finding centriole genes with Naegleria
used proteomic approaches to attempt to identify a complete parts
list for centrioles (Andersen et al., 2003; Keller et al., 2005; Kilburn
et al., 2007) or flagella (for a review, see Inglis et al., 2006).
Theoretically, proteomic analyses can identify all stably localized
proteins, including those that are species specific or required for
unrelated biological functions. By contrast, our analysis has
identified comprehensive sets of genes required specifically for
centriole and flagellar function, independent of their localization.
Of eight previously uncharacterized genes that we predict are
involved in centriole assembly, at least three have human orthologs
with a centrosome-related localization. The conservation of these
proteins in both Naegleria and human, probably spanning over a
billion years of eukaryotic evolution (Brinkmann and Philippe,
2007), indicates that these proteins are important for centriole
function. Our study adds significantly to the list of conserved
centriole components, including an additional protein (Friggin)
that is apparently specific to mature centrioles.
Our analyses extend previous observations (Fulton et al., 1995;
Levy et al., 1998) of two major programs of transcription during
Naegleria differentiation: an early round of transcription of basal
body genes and a later round of flagellar genes (Fig. 2), a timing
that mirrors the assembly of basal bodies before flagella (Fig. 1).
Although we have limited our analysis to centriole and flagella
genes, this represents only one aspect of the Naegleria amoebato-flagellate transition. A cursory analysis indicates that genes
from other core pathways, including basic metabolism and the
stress response, are also regulated differentially. We have
deposited our microarray data in the NCBI Gene Expression
Omnibus (Edgar et al., 2002) under GEO Series accession number
GSE21527 and encourage other scientists to take advantage of
this rich data set.
Materials and Methods
Naegleria differentiation and RNA isolation
N. gruberi strain NEG grown on Klebsiella was differentiated three separate times
using standard protocols (Fulton, 1970). Synchrony was estimated by counting the
percentage of flagellates after fixing in Lugol’s iodine (Fulton and Dingle, 1967),
using a phase-contrast microscope with a ⫻40 objective (n>100 for each timepoint). 107 cells were harvested at each time-point, and RNA extracted using Trizol
reagent (Invitrogen), purified using RNAeasy (Qiagen), treated with Turbo DNAse
(Ambion), and repurified with RNAeasy (Qiagen), according to the manufacturers’
instructions. RNA purity was verified by means of gel electrophoresis (supplementary
material Fig. S1) and spectrophotometry.
NimbleGen expression oligoarrays
The N. gruberi whole-genome expression oligoarray version 1.0 (NimbleGen
Systems) comprises 182,813 probe sets corresponding to 15,777 gene models
predicted on the N. gruberi genome sequence version 1.0 (Fritz-Laylin et al.,
2010a), and an additional 963 open reading frames (ORFs) identified in intergenic
regions. For each gene, 11 unique 60-mer oligonucleotide probes were designed
by NimbleGen Systems. The Naegleria V1.0 oligoarray is fully described at the
Gene Expression Omnibus (GEO) (Edgar et al., 2002) under accession number
GSE21527.
Preparation of samples, hybridization and scanning were performed by NimbleGen
Systems (Madison, WI), following their standard operating protocol. The raw data
were subjected to robust multi-array analysis (RMA) (Irizarry et al., 2003), quantile
normalization (Bolstad et al., 2003) and background correction, as implemented
in the NimbleScan software package, version 2.4.27 (Roche NimbleGen).
Reproducibility between biological replicates was inspected using MA [log-intensity
ratios (M) versus log-intensity averages (A)] and scatter plots of log intensities
(supplementary material Fig. S1), and P-values were calculated in a simple paireddata comparison model and were corrected for multiple testing using the BH (false
discovery rate controlled) procedure, all within the R statistical package (http://www.rproject.org/).
Expression clustering
The log-transformed expression data for the 310 cytoskeleton-related genes were
subjected to gene normalization followed by hierarchical clustering, with centered
correlation and complete linkage in the Cluster program (Eisen et al., 1998).
4029
Proteomics of Naegleria flagella
Flagella were isolated using published methods (Kowit and Fulton, 1974) and mass
spectrometry performed by the Vincent J. Coates Proteomics/Mass Spectrometry
Laboratory at UC Berkeley, CA. A nano LC column was packed in a glass capillary,
of 100 m inner diameter, with an emitter tip. The column comprised 10 cm of
Polaris c18 5-m packing material (Varian), followed by 4 cm of Partisphere 5 SCX
(Whatman). The column was loaded using a pressure bomb and washed extensively
with buffer A [5% acetonitrile, 0.02% heptaflurobutyric acid (HBFA)], then directly
coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL
linear ion-trap mass spectrometer. An Agilent 1200 HPLC delivering a flow rate of
30 nl/min was used for chromatography. Peptides were eluted using a 14-step
MudPIT procedure (Washburn et al., 2001), using buffer A, buffer B (80% acetonitrile,
0.02% HBFA), buffer C (250 mM ammonium acetate, 5% acetonitrile, 0.02%
HBFA) and buffer D (250 mM ammonium acetate, 5% acetonitrile, 0.02% HBFA,
500 mM ammonium acetate). The programs SEQUEST and DTASELECT were
used to identify peptides and proteins from the Naegleria genome (Eng et al., 1994;
Tabb et al., 2002).
Localization of pCCCs
The following mammalian cDNAs from the human ORF collection in the form of
Gateway entry vectors were purchased from Open Biosystems (Rual et al., 2004):
B9D2 (CV025994), MOT52 (FOR20; EL735575), POC16 (EL737049), FM14
(EL735863), SSA3 (EL735155), MOT39 (CV023936) and TECT3 (EL736819), and
these were verified by sequencing. The following cDNAs (and corresponding
accession numbers) were obtained from Open Biosystems: POC11 (BC006444),
MOT37 (BC016439) and LRRC6 (BC047286). Each ORF was amplified (primer
sequences available upon request), transferred into a Gateway donor vector
(pDONR221) and verified by sequencing. All ORFs were then transferred into the
C-terminal (pCDNA-DEST47) and N-terminal (pCDNA-DEST53) GFP-tagged
Gateway Vectors according to the manufacturers’ protocols.
A total of 20,000 U2OS cells (ATCC catalog number HTB-96) was inoculated in
0.5 ml medium [DMEM (GIBCO catalog number 10569) supplemented with 10%
FBS, 1% nonessential amino acids, and 1% sodium pyruvate] in 24-well plates
containing coverslips. Cells were transfected the next day with lipofectamine 2000,
and 14 hours later fixed for three minutes with methanol and prepared for
immunofluorescence using standard methods (http://mitchison.med.harvard.edu/
protocols/gen1.html), and the following antibodies: monoclonal antibody 20H5
against centrin (antibody 20H5) (Sanders and Salisbury, 1994), used at 1:400,
antibody CD1B4 against cenexin (antibody CD1B4) (Lange and Gull, 1995) used at
1:3, and mouse monoclonal antibody against GFP (catalog number 11814460001,
Roche) at a 1:500 dilution. Alexa-Fluor-conjugated secondary antibodies were
sourced from Invitrogen (Carlsbad, CA) and used at a 1:500 dilution.
Fluorescence deconvolution microscopy
Images were collected with SoftWorX image acquisition software (Applied Precision,
Issaquah, WA) on an Olympus IX70 wide-field inverted fluorescence microscope
with an Olympus PlanApo ⫻100, (NA 1.35), oil-immersion objective and
Photometrics CCD CH350 camera (Roper Scientific, Tuscon, AZ). Image stacks
were deconvolved with the SoftWorX deconvolution software and flattened as
maximum projections (Applied Precision, Issaquah, WA).
Sequence analysis
Domain predictions were performed using default parameters and version 24.0 of
the Pfam database (Finn et al., 2008). pCCC orthologs were collected from the
nonredundant (‘nr’) database at NCBI (Benson et al., 2009) with BLAST (Altschul
et al., 1990).
We thank Lori Kohlstaedt and the Vincent J. Coates Proteomics/Mass
Spectrometry Laboratory at UC Berkeley, CA, for help with mass
spectrometry experiments, Lani Keller, Juliette Azimzadeh, Hellen
Dawe and Tim Stearns for helpful advice, Pierre Gönczy, Rebecca
Heald, Scott Dawson, and Chandler Fulton for valuable comments on
the manuscript and Keith Gull for the generous gift of the antibody
against cenexin. This study was supported by a grant from the
University of California Cancer Research Coordinating Committee
(CRCC) to W.Z.C. and NIH grant A1054693 to W.Z.C. Article
deposited in PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/23/4024/DC1
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